In the first round of DBTL, we utilized kynureninase (KYNase), a canine uricase derived from Pseudomonas fluorescens. KYNase is a PLP-dependent enzyme, a member of the transaminase or α family IV subgroup, which is the largest family of PLP-dependent enzymes, with a monomeric molecular weight of approximately 45 kDa. As a characteristic of the transaminase family, KYNase monomers consist of a large domain and a small domain, with the active site located in the cleft between the domains at the subunit interface, containing residues from both subunits. KYNase exhibits excellent catalytic activity toward kynurenine (KYN), effectively eliminating KYN from the tumor microenvironment (TME). We obtained its sequence and sent it to a company for gene synthesis, resulting in a usable KYNase gene sequence. To ensure sustained high-level expression of our therapeutic protein, we selected Escherichia coli BL21(DE3) as the host cell. To enable long-term efficient expression of KYNase, aiming to reduce KYN concentration and improve tumor immune suppression, we selected promoter J23119 as the regulatory element.
Based on these design principles, we designed and constructed the plasmid pET29a-KYNase. We then utilized homologous recombination to integrate and construct this plasmid. We transformed it into DH5α and, after inoculating several E. coli single colonies from the transformation plates, extracted the recombinant plasmid, and performed bacterial PCR validation. Bacterial PCR confirmed the correct position of the target fragment on the gel. We then transformed the plasmid into E. coli BL21(DE3).
We validated the high-level expression of KY Nase through SDS-PAGE and Western Blot experiments.
After consulting with relevant experts, we learned that while constitutive expression can produce large amounts of protein, its high expression may affect non-tumor tissues. The experts recommended designing inducible promoters tailored to the tumor microenvironment.
For the second DBTL, Gewei Zhang interviewed Professor Jingning Zhu, an expert in molecular biology. According to the professor's advice, we decided to use the ALPaGA response promoter as a regulatory element. It will trigger the expression of engineered strains in a high-lactic-acid and hypoxia environment. It is induced at a fixed point in the tumor environment, and therapeutic substances are expressed.
The replacement of the promoter can ensure that our gene line is turned on in the tumor microenvironment and avoid the accidental expression of engineered bacteria. In order to verify whether the ALPaGA promoter can function normally under high lactic acid conditions, we constructed the recombinant plasmid pET29a-ALPaGA-eGFP and introduced it into BL21(DE3) to build the strain BL21-pET29a-ALPaGA-eGFP. Measure the expression of eGFP (green fluorescent protein) of the strain under different lactic acid concentrations by enzyme labeling to verify the best induction conditions for ALPaGA promoters.
As shown in the figure, we then designed and constructed the plasmid pET29a-ALPaGA-KY Nase, and constructed the plasmid by homologous recombination integration. We transferred it to DH5α and extracted the recombinant plasmid after picking E. coli single colony grafting bacteria on several conversion plates, and carried out colony PCR verification. The correct position of the target fragment electrophoresis band was verified by colony PCR. We transferred the plasmid into E.coli BL21(DE3) and successfully constructed the colony PCR verification BL21 (DE3)-pET29a-ALPaGA-KYNase transformation strain.
After the plasmid construction was completed, we determined the optimal lactic acid induction concentration of ALPaGA promoters. We induced protein expression at this concentration, and verified a large number of expressions of KY Nase through the Western Blot experiment.
After discussing with relevant experts, we learned that the natural KY Nase can improve enzyme activity through enzyme modification and obtain better catalysis and treatment. We decided to conduct enzyme modification experiments in the next round of DBTL.
In the third round of DBTL, we hope to enhance the enzymatic activity of KYNase through enzyme modification in order to achieve better therapeutic effects. The degradation rate of KYN is closely related to the catalytic activity of KYNase. Therefore, enhancing the catalytic activity of KYNase is of great significance for the treatment of tumors through the degradation of KYN.
First, we predicted the protein structure through Alphafold, and then used the FoldX software to mutate amino acids to alanine in sequence. We compared the changes in binding energy at each mutation site before and after the mutation. We have screened out several sites with the greatest reduction in binding energy. Primers can be designed for these sites to perform site-specific mutagenesis. Test whether the activity of the enzyme can be enhanced through experiments.
We have screened out several sites with the greatest reduction in binding energy. Primers can be designed for these sites to perform site-directed mutagenesis. Test whether the activity of the enzyme can be enhanced through experiments.
To better test the effect of enzyme modification, we compared the enzyme activity energy of the unmodified KY Nase with that of the modified KY Nase. The initial concentration of kynurenine was 4.5mM. After adding the same amount of enzyme, the absorbance of the solution was measured every ten minutes using an enzyme-linked immunosorbent assay (ELISA) reader to determine the catalytic effect of the enzyme. As shown in the figure, the catalytic efficiency of the DA33A Mutant has been significantly improved, and the effect of enzyme activity enhancement is good.
The enzyme modification effect is good, and it indicates that site 33 is very important. We plan to perform saturation mutation on this site in the future to attempt to obtain a better mutant.
We designed a lysis module based on the lysis protein PhiX174E to assist in the release of KY Nase. We hope that KY Nase produced within cells can be released into the tumor microenvironment at the appropriate time and concentration. PhiX174E is a protein encoded by the E gene of the phage PhiX174. It triggers cell lysis through a proton-dependent kinetic step by binding to the host cell membrane and oligomerizing.
We used a lactate-inducible ALPaGA promoter to control the expression of PhiX174E, hoping that when KY Nase accumulated to a certain amount, the engineered bacteria would undergo autolysis, thereby releasing our KY Nase produced intracellularly into the tumor microenvironment for treatment. Based on these design principles, we designed and constructed the plasmid pET29a-ALPaGA-RBS-PhiX174E-T7.
Due to time and experimental level limitations, we will test the cracking module in the future.
We designed and tested the lysis module and reported the project to the National Medical Products Administration (NMPA) to obtain recommendations for the practical application of the drug. Although no drugs related to synthetic biology have been approved yet, they still believe that our design has a bright future. In terms of biosafety, they recommended that we add a module to ensure that the engineered strains are completely eliminated in the external environment, thereby preventing environmental contamination. As a result, we have decided to develop a suicide switch in the next DBTL to further enhance biosafety measures.
Following discussions with officials from the National Medical Products Administration (NMPA), we designed a blue light-induced suicide switch to enable engineered bacteria to self-destruct when released into the environment (natural light), thereby safeguarding biosecurity and preventing genetic contamination. We selected the pDawn-MzaF safety module, which can activate downstream gene expression under natural light or a single blue light source. pDawn is a blue light-induced promoter that can activate downstream gene expression under natural light or a single blue light source. MazF is an RNAase that efficiently degrades bacterial mRNA, thereby inhibiting bacterial growth and reproduction.
We designed the plasmid pET29-pDawn-MazF using SnapGene software to achieve a suicidal effect. Due to time constraints, we only completed the design of the plasmid and plan to test its performance in the future.
Due to time constraints, we have only completed the design of the plasmid and will test its effectiveness in the future.
Through discussions with officials from the NMPA, we recognized the importance of biomedical contamination. Therefore, we designed a suicide switch to prevent such issues and hope that this module will contribute to improving biosafety. In the future, we will complete the unfinished experiments and integrate all modules to build a complete KINETiC therapy.
